Partially modified and retro-inverted tetrapeptides analogues of C-reactive protein fragments

ABSTRACT

Retro-inverted tetrapeptides of formula I ##STR1## wherein R is a hydrogen atom or the side-chain of threonine; R 1  is the side-chain of arginine, leucine or glutamine; and R 2  is a hydrogen atom or a metabolically perishable acyl group; with the proviso that when R 1  is the side-chain of arginine, R cannot be the side-chain of threonine; diastereo-isomeric forms and pharmacologically acceptable salts, esters and amides thereof. These compounds are useful as immuno-stimulating agents.

The present invention relates to retro-inverted tetrapeptides analoguesof C-reactive protein fragments (hereinafter CRP).

CRP is a protein generally having very low blood concentration, whichrises up to two thousand times following inflammatory process [J. J.Morley and I. Kushner, Am. N.Y. Acad. Sci., 389, 406-418 (1989)]. F. A.Robey et al , J. Biol. Chem., 262 No.15 7053-7057 (1987) disclose threeCRP tetrapeptide sequences very similar to the ones of tuftsin. Thechemically sinthetised tetrapeptides show to stimulate the phagocyticleukocytes, to produce superoxide and to induce mononuclear cells toproduce interleukin 1, in a qualitatively and quantitativelytuftsin-like manner. Like tuftsin, the three CRP tetrapeptides should berapidly in vivo metabolised by proteases, and yield peptide metaboliteswhich could competitively inhibit the biological activity/ies of theparent peptides. It has been now surprisingly found that partiallymodified and N-terminal retro-inverted analogues of said CRPtetrapeptide fragments show not only a considerable stability againstthe enzymatic degradation while maintaining the immunomodulatingactivity already seen for tuftsin (see EP-A-0 253 190), but inparticular they are able to determine different biological effectsdepending on the structure and the dose used, specifically in thetreatment of septic shock. Therefore the present invention relates toretro-inverted tetrapeptides of the general formula I ##STR2## wherein Ris a hydrogen atom or the side-chain of threonine; R₁ is the side-chainof arginine, leucine or glutamine; and R₂ is a hydrogen atom or ametabolically perishable acyl group; with the proviso that when R₁ isthe side-chain of arginine, R cannot be the side-chain of threonine; andtheir diastereoisomeric forms and pharmacologically acceptable salts,esters and amides.

Particularly, the invention relates to the retro-inverted tetrapeptidesgGly-(R,S)mLys-Pro-Arg, gThr-(R,S)mLys-Pro-Leu, gThr-(R,S)mLys-Pro-Gln,wherein the prefixes g and m mean that the aminoacid is, respectively, agem-diamine and malonyl residue, and the diastereoisomeric formsthereof.

The retro-inverted tetrapeptides of the present invention aresynthetised in accordance with known methods, which the skilled in theart may choose depending on the kind of aminoacids to retroinvert.

For example, when the gem-diamine residue is the 1,1-diaminomethanegroup (gGly), the retro-inverted tetrapeptide may be prepared by firstreacting the Meldrum's acid derivative c-mLys(Z) (i.e.5-[4-benzyloxycarbonyl]-2,2-dimethyl-1,3-dioxane-4,6-dione) withH-Gly-NH₂ in the presence of a sylanising agent such asN,O-bis(trimethylsylyl)acetamide (TSMAc), trimethylsylylchloride(TMS-Cl) or trimethylsylylcianide (TMSCN) (M.J.O. Anteunis & Char. Becu,Bull. Soc. Chim. Belg., 96, 119-139 1986). The second group on themalonyl residue of the pseudopeptide OH-(R,S)mLys(Z)-Gly-NH₂(Z=benzyloxycarbonyl) thus obtained is condensed with t-butyl prolinatein the presence of dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBT) to give the pseudotripeptide[(R,S)mLys(Z)-Gly-NH₂ ]-Pro-H. Its proline carboxy group is activatedwith DCC and N-hydroxy-succinimide (HOSu) and reacted with unprotectedarginine [Gottlieb, P. et al., Ann. N.Y. Acad. Sci., 419, 12 (1983 )] toobtain the retro-inverted tetrapeptide [(R,S)mLys(Z)-Gly-NH₂]-Pro-Arg-OH. The purification is then effected through RP-DCdeplacement chromatography, and by this way it is also possible to yieldthe separation of diastereoisomers, if desired. The purified product iscatalytically hydrogenated by HCOOH in the presence of palladium, toremove the remaining protective groups, and then treated with[I,I-bis(trifluoroacetoxy)-iodo]benzene (TIB) for turning the glycinecarboxamide into the N-terminal residue of gGly. One last purificationby ion exchange chromatography enable to obtain the final product asacetate.

Another example is where threonine is the gem-diamine residue. Thesynthesis of the retro-inverted tetrapeptide starts from a C-terminaldipeptide obtained through condensation of Z-Pro-OH and H-Y-OtBu,wherein Y represents the aminoacid Leu or Gln in accordance with whatsaid in the definition of formula I, optionally suitably protected, andthrough subsequent removal of the benzyloxycarbonyl group by catalytichydrogenation. Such dipeptide is reacted with the N-terminalretro-inverted dipeptide prepared, as described, for example, in theItalian patent application No. 21349 A/90.

MNP-Thr(tBu)-OH obtained by acylating previously sylanised H-Thr(tBu)-OHwith MNP-COOH, in the presence of suitable carboxy-activating agents, istreated with biphenylphosphorazide (DPPA) yielding the relevant azide,which gives the isocianate by heating. This is treated with tiophenol inthe presence of catalytic amounts of amine, preferably a tertiary aminesuch as triethylamine or diisopropylamine, to obtain thephenylthiocarbonyl derivative MNP-gThr(tBu)-PTC. Subsequently the amineresidue is deprotected, for example, by treatment with diluted sodiumhydroxide to give MNP-gThr(tBu)-H. The condensation of this latter withc-mLys(Boc) gives the N-terminal pseudodipeptideMNP-gThr(tBu)-(R,S)mLys(Boc) which, in its turn, is condensed withH-Pro-Y-OtBu, wherein Y is as defined above, in the presence of DCC/HOBTto give the protected retro-inverted tetrapeptide of formula I wherein Ris 1-hydroxyethyl. By means of treatment with an acid, all theprotective groups are removed except for the one of the gem-diamineresidue. At the same time as the purification via RP-DC, it is possibleto separate the two diastereoisomers, if desired. The MNP group may beremoved through catalytic hydrogenation, for example, by using ammoniumformate on sponge Pd. The retro-inverted tetrapeptide is submitted tothe final purification by suitable chromatographic methods.

Preparative examples of some retro-inverted tetrapeptides representativeof the claimed class are herein provided, which are not to be intendedas limitating the invention in any way.

The HPLC analysis of the aminoacid derivatives, of the protectedfragments and of the retro-inverted tetrapeptide is carried out underthe following experimental conditions:

column: Lichrosorb RP-18;

flow: 1.5 ml/min;

detector: Merck L-4200 UV-VIS (230 or 254 nm);

eluent A: water 90%, MeCN 10%, trifluoroacetic acid (TFA) 0.1%;

eluent B: MeCN, TFA 0.1%;

eluent C: water, TFA 0.1%;

Gradients:

(I): from 0 to 40% B in A (20'), to 80% B in A (10')

(II): from 37 to 80% B in A (20')

(III): from 0 to 50% A in C (20'), to 100% A (3'), to 40% B in A (20')

(IV): from 10 to 40% A in C (8'), to 100% A (2'), to 40% B in A (20')

In the ion exchange purifications, the FPLC system of Pharmacia equippedwith LKB UVICORD S II detector with filter at 226 nm, Pharmacia recorder(chart speed=0.1 cm/min) and Pharmacia FRAC 200 fraction collector isemployed.

The aminoacid and NH₃ compositions and ratios (as specific datum of thegem-diamino residue) of the various retro-inverted peptides aredetermined by Beckman SYSTEM GOLD automatic aminoacid analyzer afterhydrolysis with HCl 6M at 110° C. for 22 hours.

The alphanumeric code in bracket after the name of some compounds is aninternal code of indentification.

EXAMPLE 1

H-gGly-(R,S)mLys-Pro-Arg-OH.2AcOH (ITF 1127)

A) A suspension of H-Gly-NH₂.HCl (3.32 g, 30 mmoles) in THF (200 ml) wasadded with TMSAc (19.6 ml, 80 mmoles) and TMS-Cl (2.54 ml, 20 mmoles) insequence. After 30 minutes c-mLys(Z) (6.98 g, 20 mmoles) was added, andthe reaction mixture was stirred at room temperature for further 3hours. The solvent was evaporated under vacuum and the residue was takenup with water (200 ml) while adjusting the pH to 3 with HCl 0.1N. After1 hour under stirring at room temperature, the formed solid wasfiltered, washed with water, and dissolved in hot ethanol. The solidobtained after cooling was filtered, washed with ethyl ether and dried.3.8 g of (R,S)mLys(Z)-Gly-NH₂ were obtained (yield: 51%).

HPLC [gradient (I) (254 nm)]: r.t. 13.5 minutes; purity 98%; m.p.: 161°C. (dec.)

The ¹ H-NMR confirmed the structure of the product.

B) A solution of the product under A) (3.65 g, 10 mmoles) in DMF (15ml), cooled in iced bath, was added with HOBT (1.43 g, 10.5 mmoles) andDCC (1.96 g, 9.5 mmoles) in sequence. After 30 minutes under stirring,the mixture was filtered and the filtrate was added to a solution of DMF(10 ml) containing HCl.H-Pro-OtBu (2.49 g, 12 mmoles) and TEA (1.67 ml,12 mmoles). The mixture was stirred for 3 hours, then the solvent wasevaporated under vacuum, the residue was taken up with ethyl acetate (50ml) , and the organic phase was washed with potassium bisulphate 2%,sodium hydrocarbonate 5%, and neutralised with water, then anhydrifiedon sodium sulphate. The obtained 4.2 g of a light oil (yield: 82%) weredissolved in a mixture of DMC and TFA (1:1 v/v, 16 ml) and stirred for 1hour. The solvent was evaporated under vacuum, and the residue wasground with ethyl acetate and ethyl ether to obtain 3.6 g of[(R,S)mLys(Z)-Gly-NH₂ ]-Pro-OH as white solid (yield: 79%).

HPLC [gradient (I) (254 nm)]: r.t. 14.6 and 15.7 minutes (twodiastereoisomers); purity 97%.

The ¹ H-NMR confirmed the structure of the product.

C) A solution of the product under B) (3.36 g, 7.5 mmoles) in THF (50ml), was added with HOSu (0.95 g, 8.25 mmoles) and, after cooling to-10° C., with DCC (1.54 g, 7.5 mmoles). After 4 hours under stirring atroom temperature, the reaction mixture was filtered and the filtrateadded to a solution of DMF/water (7:3 v/v, 125 ml) containing H-Arg-OH(1.74 g, 10 mmoles) and KCl (0.74 g, 10 mmoles). The reaction mixturewas kept under stirring for 90 minutes at room temperature, then thesolvent was evaporated under vacuum, and the residue was washed sometimes with ethyl ether and dried. The solid obtained was dissolved in anaqueous solution of TFA (0.16 v/v, 30 ml) and purified through RP-DC inthree aliquots. In each purification 10 ml of solution were charged on aDynamax 300 Å C₁₈ (21.4×300 mm) column previously balanced with watercontaining TFA (0.1% v/v), at a flow of 2.7 ml/minute. At the end of thecharging the column was eluted, still at 2.7 ml/minute, with .an aqueoussolution 50 mM of benzyldimethylhexadecylammonium chloride (BDHA-Cl)containing TFA (0.1% v/v). After about 1 hour of elution, 2.7 mlfractions were collected. The fractions were analyzed by HPLC [gradient(I)], and the ones containing impurities were eliminated, while theothers were collected in three groups respectively containing isomer A,the mixture of the two isomers, and isomer B of the compound[mLys(Z)-Gly-NH₂ ]-Pro-Arg-OH. At the end of the three chromatographies,the three groups of fractions were freeze-dried yielding 1.9 g of isomerA, 0.45 g of the isomeric mixture and 1.8 g of isomer B (total yield:89%).

HPLC [gradient (I) (220 nm)]: r.t. 13.6 minutes (isomer A), purity: 96%;r.t. 14.7 minutes (isomer B); purity 93%+4% of isomer A.

FAB-MS: m/z=619 amu [M+H]⁺ ; m/z=485 amu [M-Z+H⁺ (identical spectra forthe two isomers).

D) Fresh sponge Pd (about 0.1 g) was added to a solution of the isomermixture under C) (0.31 g, 0.5 mmoles) in formic acid (85%, 5 ml), andthe mixture was slowly stirred at room temperature for 90 minutes. Afterfiltering off the catalyzer, the solvent was evaporated under vacuum andthe residue taken up with water and freeze-dried. The solid obtained wasdissolved in a mixture of DMF/water (3:1 v/v, 5 ml) and added with TIB(0.43 g, 1 mmole) . The reaction mixture, sheltered from the light, wasstirred at room temperature for 16 hours. The solvent was evaporatedunder vacuum and the residue dissolved in water, washed with ethyl etherand freeze-dried. The solid obtained was dissolved in water at pH 6 andcharged (flow=1 ml/minute) on a column (6×200 mm) filled with CM-52carboxymethylcellulose and previously balanced with a solution ofammonium acetate 15 mM at pH 6. After charging, the column was elutedwith a linear gradient of ammonium acetate from 0.15 mM to 150 mM within6 hours, at a flow of 1 ml/minute, while maintaining pH at 6. Fractionsof 3 ml were collected and analyzed by HPLC [gradient (III)]: thefractions contaning the product in title (ITF 1127) were collected andfreeze-dried more times. The solid obtained was dissolved in absoluteethanol and 0.085 g of product precipitated by adding ethyl ether toobtain (yield: 30%).

HPLC: [gradient (III)]: r.t. 8.8 (isomer A) and 10.2 (isomer B) minutes;purity 99%.

FAB-MS: m/z=457 amu [M+H]⁺

Following an analogous procedure 0.425 g was obtained (yield: 30%) ofisomer A (ITF 1357)

¹ H-NMR (200 MHz; DMSO)

t (1H; NHG) 8.25; d (1H; NH-R) 7.20; m (1H; C.sub.α P) 4.30; m (4H; αG;αR; δ_(A) P) 3.93÷3.64; m (3H; δ_(B) P; δK) 3.61÷3.37; t (2H; δR) 3.04;t (2H; εK) 2.74; m (6H; βP; τP; βK) 2.09÷1.79; s (6H CH₃ COOH) 1.75; m(8H; δK; τK; βR; τR) 1.70÷1.18.

Following the procedure for isomer A it was obtained 0.45 g of productas isomer B (ITF 1358) (yield: 32%) and 0.1 g of mixture (ITF 1127).

HPLC: [gradient (III)]: 10.2 minutes; purity 96%+isomero A.

FAB-MS: m/z=457 amu [M+H]

¹ H-NMR (200 MHz; DMSO)

t (1H; NGH) 8.30; d (0.4H; NHR) 7.57; d (0.6H; NHR) 7.47; m (1H; C.sub.αP) 4.43; m (3H; αG; αR) 3.97÷3.82; m (4H; δP; δK) 3.63÷3.33; m (2H; δR)3.06; m (2H; εK) 2.72; s (6H CH₃ COO⁻) 1.76; m (14H; β,τP; β,τ,δK; β,τR)1.21÷1.15.

EXAMPLE 2

H-gThr-R,S)mLys-Pro-LeU-OH.AcOH (ITF 1192)

A) A solution of bis(trichloromethyl)carbonate (3.71 g, 12.5 mmoles) inmethylene chloride (125 ml) at 0° C. was added with 1-methylimidazole(5.96 ml, 75 mmoles) in 80 ml of methylene chloride. After 5 minutesMNP-OH (5.63 g, 25 mmoles) dissolved in methylene chloride (80 ml) wasadded and, after further 5 minutes, it was followed by H-Thr(tBu)-OH(5.26 g, 30 mmoles) previously sylanized with TMSCN (11.3 ml, 90mmoles), and further 200 ml of methylene chloride. After 10 minutes, thereaction mixture was washed with an aqueous buffer acidified to pH 3.5,anhydrified and evaporated to dryness. The residue taken up with sodiumcarbonate 5% (300 ml) was washed with methylene chloride. The aqueousphase was added with further 200 ml of ethyl acetate and the pH broughtto 5.7. The organic phase was separated and anhydrified, and the solventevaporated to obtain MNP-Thr(tBu)-OH as an oil (6.5 g, yield: 68%).

HPLC [gradient (I) (254 nm)]: r.t. 25.1 minutes; purity: 88%.

B) A solution of the compound under A) (5.2 g, 13.6 mmoles) in anhydroustoluene (50 ml) at 0° C. was added with DPPA (3.22 ml, 14.9 mmoles) andTEA (2.09 ml, 14.9 mmoles). After 4 hours the reaction mixture waswashed three times with a solution saturated with sodium hydrocarbonateand with a solution saturated with sodium chloride, both the solutionsbeing pre-cooled to 0° C., then it was anhydrified. The solutioncontaining the azide was heated to 80° C. and maintained at suchtemperature for 40 minutes. The formed isocianate was added withtiophenole (1.39 ml, 13.6 mmoles) and a catalytic amount of TEA (191 ul,1.36 mmoles), at room temperature. After one night, the mixture wastreated with ethyl acetate and water, and the organic phase was washedwith potassium bisulphate 2%, sodium hydrocarbonate 5% and neutralisedwith water, then anhydrified on sodium sulphate, evaporated to drynessand the oil obtained was ground with petroleum ether. There wereobtained 4.7 g of MNP-gThr(tBu)-PCT (yield: 71.2%). HPLC [gradient (II)(254 nm)]: r.t. 14.9 minutes; purity: 95%.

C) A mixture of sodium hydroxide (1M, 26 ml) and water 877 ml) maintanedat 0° C. was slowly added with a solution of the compound under B) (4.7g, 9.6 mmoles) in THF (64 ml). After 5 minutes from the end of theaddition, the reaction mixture was neutralised with HCl (1M, 26 ml). TheTHF was evaporated, and chloroform (80 ml) was added and the pH of thebiphasic mixture was brought to 9.0 with sodium hydroxide 1M. Theorganic phase was separated off and the aqueous one was treated withchloroform three times, while adjusting the pH to 9.0 each time: theorganic phases were collected and evaporated to dryness. The residueresuspended in ethyl ether was added with water (50 ml) and the mixturewas titrated to pH 3.5 with HCl 1M until constant pH. The aqueous phasewas separated and the treatment repeated twice. The collected aqueousphases were freeze-dried to obtain 2.8 g of MNP-gThr(tBu)-H.HCl (yield:85%).

HPLC [gradient (I) (254 nm)]: r.t. 16.43 minutes; purity: 99.9%.

D) A solution of the compound under C) (2.6 g, 6.6 mmoles) andc-mLys(Boc) (2.4 g, 8 mmoles) in THF (120 ml) was slowly added withTMSAc (4.6 ml, 20 mmoles). After 20 hours the reaction mixture wasevaporated and the residue taken up with water and brought to pH 3.5with HCl 1M in the presence of ethyl acetate. The aqueous phase wasestracted three times with ethyl acetate, and the collected organicphases were anhydrified, reduced to a small volume, and added withdiisopropyl ether to give 3.4 g of MNP-gThr(tBu)-(R,S)mLys(Boc)-OH(yield: 85%).

HPLC [gradient (II) (230 nm)]: r.t. 9.9 and 10.2 minutes (twodiastereoisomers); purity 93%.

E) A solution of Z-Pro-OH (2.4 g, 10 mmoles) and H-Leu-OtBu.HCl (2.7 g,12 mmoles) in methylene chloride (50 ml) was added with TEA (3 ml, 22mmoles) followed by BOP (4.4 g, 10 mmoles) and HOBT (1.35 g, 10 mmoles). After 5 hours the reaction mixture was treated once with a solutionsaturated of sodium chloride, then evaporated to dryness. The residuewas divided between ethyl acetate and water. The organic phase waswashed with potassium bisulphate 2%, sodium hydrocarbonate 5%,neutralised with water and anhydrified on sodium sulphate, then dried,and from the residue dissolved in diisopropyl ether 3.8 g ofZ-Pro-Leu-OtBu were obtained by addition of petroleum ether (yield:92.7%).

HPLC [gradient (I) (230 nm)]: r.t. 28.6 minutes; purity 100%.

F) The dipeptide under E) (1.5 g, 3.5 mmoles) was dissolved in methanol(70 ml) and this solution, under nitrogen, was added with Pd/C catalyzer(100 mg) and then, very slowly, with triethylsylane (2.9 ml, 18 mmoles).After 3 hours the reaction mixture was filtered and the solventevaporated, thus obtaining 1 g of H-Pro-Leu-OtBu (yield: 100%).

HPLC [gradient (I) (230 nm)]: r.t. 15.9 minutes; purity 93%.

G) A solution of the pseudodipeptide under D) (1.1 g, 1.9 mmoles) inmethylene chloride (15 ml) was added with HOBT (320 mg, 2.3 mmoles)dissolved in 200 ml of DMF. The temperature was brought to 0° C. andDCCI (392 mg, 1.9 mmoles) was added. After 15 minutes the mixture wasfiltered in a flask containing the C-terminal dipeptide under E) (540mg, 1.9 mmoles) and left to react overnight. After evaporating thesolvent, the residue was divided between ethyl acetate and water, andthe organic phase was washed with potassium bisulphate 2%, sodiumhydrocarbonate 5% and neutralised with water, then anhydrified on sodiumsulphate. From the dried organic phase by grinding of the residue withdiisopropyl ether 12.3 g of MNP-gThr(tBu)-(R,S)mLys(Boc)-Pro-Leu-OtBwere obtained (yield: 77%).

HPLC: isocratic from 65% of B (230 and 254 nm): r.t. 8.2 and 8.6minutes; purity: 100%.

H) An aliquot of the compound under G) (1.4 g, 1.3 mmoles) was treatedin iced bath with concentrated HCl (5 ml) for 8 minutes. The reactionmixture was evaporated to dryness, then taken up with water andfreeze-dried. There were obtained 910 mg of a crude that was purifiedthrough RP-DC on a Dynamax column (300 Å C18, 12 um, 21×250 mm)employing benzyl-dimethyldodecylammonium bromide (BDDA-Br, 50 mM in 90%of water, 10% of acetonitrile and 0.1% of TFA) as displacer, at a flowof 2.7 ml/minute. Thus three fractions of MNP-gThr-mLys-Pro-Leu-OH wererecovered: 320 mg of isomer A, 320 mg of isomer B and 160 mg ofdiastereoisomeric mixture.

HPLC [gradient (I) (230 nm)]: r.t. 16.1 and 18 minutes; purity 99.9%.

I) The isomer A under H) (315 mg, 0.4 mmoles) was dissolved in methanol(15 ml) and added with sponge Pd activated with formic acid and asolution of ammonium formate (120 mg) in formic acid (5 ml). After 2hours the catalyzer was filtered off and, after evaporation of thesolvent, the residue was taken up with water. The aqueous phase waswashed with ethyl ether and freeze-dried yielding 200 mg of a crude thatwas purified by ion exchange on a Pharmacia XK16 column (16×200 mm) ofS-Sepharose Fast Flow employing ammonium acetate 0.015M, pH 6.0 (A) andammonium acetate 0.3M, pH 6.0 (B) as eluent in linear gradient from 0 to25 % of B in A (30') and then in isocratic for 40 minutes, at a flow of3 ml/minute. The fractions collected every 2 minutes were analized byHPLC and the ones containing the product in title were freeze-dried.

HPLC [gradient (III) (230 nm)]: r.t. 12.8 minutes; purity 97%.

Aminoacidic composition: Pro(1) 1.0; Leu(1) 1.0; NH₃ (2) 1.9; peptidecontent: 77 umoles (40 mg; yield: 20%).

¹ H-NMR (200 MHz; ¹ H-DMSO; 30° C.) (isomer A -ITF 1432): d (1H; NH-_(g)T) 7.79; d (1H; NH-L) 6.93; In (2H; Cα-P, Cα-_(g) T) 4.31+4.21; q (1H;Cα-L) 3.84; m (1H; Cδ-P) 3.74; In (3H; Cδ² P; Cβ-_(g) T; Cα-mK)3.63÷3.37; In (2H; Cε-mK) 2.75; m (4H; Cβ,Cτ-P) 2,19÷1,74; m (9H;Cβ,Cτ,Cδ-mK; Cβ,Cτ-L) 1.73÷1.13; d (3H; Cτ-_(g) T) 1.06; d (3H; Cδ¹ L)0.88; d (3H; Cδ² L) 0.87.

Following the same procedure applied for isomer, isomer B (ITF 1443) wasobtained:

¹ H-NMR (200 MHz; ¹ H-DMSO; 30° C.) d (0.9.; N^(A) H-_(g) T) 8.13; d(0.1H; N^(B) H-_(g) T) 8.03; d (0.1H; N^(B) H-L) 7.45; d (0.9H; N^(A)H-L) 7.36; m (2H; Cα-P, Cα-_(g) T) 4.37÷4.24; m (1H; Cα-L) 3.86; m (3H;Cδ-P; Cβ-_(g) T) 3.56÷3.38; m (1H; Cβ-mK) 3.16; m (2H; Cε-mK) 2.76; m(13H; Cβ,Cτ-P; Cβ,Cτ,Cδ-mK; Cβ,Cτ-L) 2.23÷1.15; d (3H; Cτ-_(g) T) 1.03;d (6H; Cδ-L) 0.88.

EXAMPLE 3

H-gThr-(R,S)mLys-Pro-Gln-OH.AcOH (ITF 1193)

A) Z-Gln-OH (5.6 g, 20 moles) was dissolved in glacial acetic acid (60ml) and added with Trt-OH (Trt=trityl) (10.4 g, 40 mmoles), Ac₂ O (3.77ml, 40 mmoles) and sulphuric acid. The reaction mixture was heated to50° C. and kept at this temperature for 90 minutes, then cooled to roomtemperature and treated with water. The precipitate obtained wasfiltered, suspended in water and extracted three times with ethylacetate. From the collected and anhydrified organic phases 8.12 g ofZ-Gln(Trt)-OH precipitated by concentration and addition of n-hexane(yield: 77%).

HPLC [gradient (II) (230 nm)]: r.t. 11.3 minutes; purity: 100%.

B) A solution of the product obtained under A) (4 g, 7.6 mmoles) inmethylene chloride (50 ml) was added with borotrifluoro etherate (153ul) and t-butyl-2,2,2-trichloroacetimidate (TBTA; 4.2 g, 15.3 mmoles).After 10 minutes the reaction mixture was washed once with a solutionsaturated with sodium hydrocarbonate and water, then anhydrified andevaporated to dryness. The residue obtained was dissolved in methanol(60 ml) and 3.5 g of Z-Gln(Trt)-OtBu precipitated by addition of water(yield: 81%).

HPLC [gradient (II) (230 nm)]: r.t. 17.7 minutes; purity: 100%.

C) A solution of the compound under B) (2.5 g, 4.3 mmoles) in 200 ml ofmethanol, saturated with nitrogen, was added with sponge Pd activatedwith formic acid, and a solution of ammonium formate (200 mg in 5 ml).After 3 hours the catalyzer was filtered off and the methanol solutionevaporated to dryness. The residue was taken up with ethyl acetate,washed once with sodium hydrocarbonate 5% and then neutralised withwater. The organic solution was anhydrified and concentrated to smallvolume, then cooled to 0° C. and treated with 1 equivalent of HCl inethyl acetate (4M, 1.08 ml). By subsequent addition of petroleum ether 2g of HCl.H-Gln(Trt)-OtBu precipitated (yield: 100%). HPLC [gradient (II)(230 nm)]: r.t. 7.4 minutes; purity: 100%.

D) Z-Pro-OH (1.15 g, 4.6 mmoles) was dissolved in methylene chloride (15ml) and added with HOBT (0.7 g, 5.5 mmoles) in DMF (250 ul). Thissolution, in iced bath, was added with DCCI (0. 95 g, 4.6 mmoles). After15 minutes the reaction mixture was filtered in a flask containing thecompound under C) (2 g, 4.1 mmoles) and neutralised with TEA (58 ul, 4.1mmoles) . After 18 hours, the solvent evaporated, and the residue wasdivided between ethyl acetate and water, the organic phase was washedwith potassium bisulphate 2%, sodium hydro carbonate 5% and neutralisedwith water, then anhydrified on sodium sulphate. From the organicsolution concentrated to small volume 2.6 g of Z-Pro-Gln(Trt)-OtBu wereobtained by addition of n-hexane (yield: 93%).

HPLC [gradient (II) (230 nm)]: r.t. 16.8 minutes; purity: 100%.

E) The protected dipeptide under D) (2.4 g, 3.5 mmoles) was hydrogenatedin 100 ml of methanol by sponge Pd and formic acid, as under C). Afterfiltering off the catalyzer and evaporating methanol, the residue wastaken up with ehtyl acetate, washed once with sodium hydrocarbonate 5%and anhydrified. The concentrated organic phase was treated with HCl inethyl acetate (4M, 875 ul) at 0° C., and 2 g of HCl.H-Pro-Gln(Trt)-OtBuwere obtained by adding petroleum ether (yield: 100%).

HPLC [gradient (II) (230 nm)]: r.t. 7.8 minutes; purity: 100%.

F) A solution of the pseudodipeptide obtained under Example 2,D) (1.38g, 2.26 mmoles) in methylene chloride (15 ml) was added with HOBT (382mg, 2.82 mmoles) dissolved in 200 ul of DMF. The temperature was broughtto 0° C. and DCCI (466 mg, 2.26 mmoles) was added. After 15 minutes themixture was directly filtered in a flask containing the dipeptide underE) (1.31 g, 2.26 mmoles), neutralised with TEA (318 ul, 2.26 mmoles) andleft to react overnight. After evaporation of the solvent, the residuewas divided between ethyl acetate and water, the organic phase waswashed with potassium bisulphate 2%, sodium hydrocarbonate 5%, andneutralised with water, then anhydrified on sodium sulphate. The organicphase was evaporated to dryness and then taken up with ethylacetate/n-hexane (1:1 v/v, 15 ml), and 2.3 g ofMPN-gThr(tBu)-(R,S)mLys(Boc)-Pro-Gln(Trt)-OtBu were obtained by additionof n-hexane (yield: 92%).

HPLC [gradient (II) (230 nm)]: r.t. 21.5 and 22.0 minutes (twodiastereoisomers); purity: 92.5%.

G) The retro-inverted tetrapeptide under F) (2.13 g, 1.87 mmoles) wasdissolved in methylene chloride/TFA (1:1 v/v, 40 ml) at roomtemperature. After 90 minutes, the solvent was evaporated, the crudeprecipitated by addition of ethyl ether. Thus 1.416 g ofMPN-gThr-(R,S)mLys-Pro-Gln-OH were obtained (yield: 95%).

HPLC [gradient (I) (230 nm)]: r.t. 12.4 minutes; purity: 91.5%.

Aminoacidic composition: Pro(1) 1.0; Gln(1) 1.0; NH₃ (3) 2.9; peptidecontent: 92.7%. The protected retro-inverted tetrapeptide was purifiedby RP-DC un a Dynamax (300 Å C18, 12 um, 21.4×300 mM) column by usingBDDA-Br (50 mM in water, 0.1% TFA) as displacer, at a flow of 2.7ml/minute. Thus 1.04 g of the product were recovered (yield: 81%).

HPLC [gradient (I) (230 nm)]: r.t. 12.4 minutes; purity: 100%. In agradient from 0 to 20% in A (20 minutes): r.t. 17.5 and 17.8 minutes(two diastereoisomers); purity: 100%.

H) The protected retro-inverted tetrapeptide under G) (875 mg, 1.1mmoles) was dissolved in 15 ml of an aqueous solution of ammoniumformate (0.25M, pH 3.0), added to sponge Pd activated with ammoniumformate (0.5M, pH 3.0), and left to react at 60° C. for 15 minutes. Thetreatment was repeated four times, raising the reaction temperature to70° C., until the HPLC showed the disappearance of the startingcompound. The catalyzer was then filtered off, and the aqueous phase waswashed with ethyl ether and freeze-dried. The crude was purified throughion exchange on a Pharmacia XK26 column (26×300 mm) of CM-Sephadex C-25by employing ammonium acetate 0.015M, pH 6.0 (A) and ammonium acetate0.3M, pH 6.0 (B) as eluent with a linear gradient from 0 to 100% of B(360 minutes, at a flow of 4 ml/minute. Fractions of 3.7 ml werecollected: there were freeze-dried the ones that had shown to containthe product in title. HPLC [gradient (IV) (230 nm)]: r.t. 6.5 and 7.7minutes (two diastereoisomers in a ratio 1:1); purity: 99%.

Aminoacidic composition: Pro(1) 1.0; Gln(1). 1.0; NH₃ (3) 2.9; peptidecontent: 966 umoles (513 mg); yield 88%. ¹ H-NMR (200 MHz; DMSO)

d (0.5H; NHT) 8.26; d (0.5H; NHT) 7.99; d (0.5H; NHQ) 7.49; s (1H; N Q)7.31; d (0.5H; NαQ) 7.04; s (1H; NαQ) 6.67; m (1H; C.sub.α T) 4.31; m(1H; C.sub.α P) 4.22; m (2H; CαQ; CδP) 3.83÷3.70; m (3H; CβT; CαK; Cδ'P)3.64÷3.34; m (2H; CτK) 2.74; m (8H; CβP; CβK; Cβ e CτQ) 2.17÷1.66; s (6HCH₃ COOH) 1.85; m (6H; CτP; Cτ e CδK) 1.64÷1.12; t (3H; CτT) 1.04.

Some compounds representative of the invention were tested for thebiological activity thereof.

Stimulation of in vitro IFN-τ production by murine splenocytes

The splenic cells were obtained from spleen of BALB/C mice as singlecells suspension. The splenocytes thus obtained were resuspended in RPMI1640 culture medium (Flow Lab., Hertz, UK) containing 5% of fetal calfserum (FCS) (HyClone, ST, Utah, USA) at a final concentrazione of 10⁷cells/ml, and incubated in 96-well plates for 24 hours at 37° C. in thepresence of the tested compounds at the indicated concentration. At theend of the incubation the supernatant was collected, filtered by 22 μmfilter and freezed to -80° C. until the time of the test carried out byELISA commercial kit (Genzyme, Boston, Mass., USA).

The results are set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                                  U/ml (Stimulation index)                                            Dose (μg/ml)                                                                           Ex.1       Ex.2       Ex.3                                        ______________________________________                                        0 (control) 31         31         41                                          0.1         33 (1.1)   57 (1.8)   60 (1.5)                                    1.0         60 (1.9)   52 (1.7)   60 (1.5)                                    10.0        49 (1.6)   60 (1.9)   68 (1.7)                                    ______________________________________                                    

The results obtained as U/ml and stimulation index (IS) with respect tothe control (Untreated cells), showed that the compounds of theinvention are able to dose-dependently stimulate IFN-τ production bymurine splenocytes. Particularly, the compound of example 1 showed to bethe most powerful one of the present class: indeed it doubled thecytokine in question already at a dose of 1.0 μg/ml.

Stimulation of IL-1 production by murine peritoneal macrophages

Murine peritoneal macrophages were obtained by inoculation of a solutionof hydrolized starch (BDH Chemicals, Poole, Dorset, UK) into theperitoneal cavity of BALB/C. mice, three days before sacrificing theanimals. The peritoneal cells were then collected by washing theperitoneal cavity with a solution of RPMI 1640 containing 10% of FCS,and resuspended in the same solution at a concetration of 10⁶ cells/mlin 96-well plates. The incubation was performed for 96 hours at 37° C.in the presence of the compounds in question. At the end of thetreatment the supernatants were collected and used for dosing cytokineby means of a suitable ELISA commerical kit (Genzyme, Boston, Mass.,USA).

The results are set forth in Table 2.

                  TABLE 2                                                         ______________________________________                                                  U/ml (Stimulation index)                                            Dose (μg/ml)                                                                           Ex.1       Ex.2       Ex.3                                        ______________________________________                                        0 (control) 15         15         15                                           0.01       6 (0.4)    90 (6.0)   150 (10.0)                                  0.1         2 (0.1)    60 (4.0)   160 (10.7)                                  1.0         2 (0.1)    47 (3.1)   155 (10.3)                                  10.0        2 (0.1)    50 (3.3)   140 (9.3)                                   ______________________________________                                    

The results obtained as U/ml and IS with respect to the control, revealthat the tested compounds show a remarkable stimulatory effect.Particularly, the compound of Example 3 increased about ten times(9.3<IS<10.7) the IL-1 production at all the doses used.

Stimulation of the nitric oxide by murine peritoneal cells

The peritoneal cells were obtained as described in the previous test,and incubated for 96 hours at 37° C. in the presence of the compounds inquestion and of lipopolysaccharide at a concentration of 30 μg/ml. Atthe end of the treatment the supernatants were collected and the nitricoxide content was determined by the chemiluminescence test described byPalmer R. M. J. et al, Nature, 327, 524, 1987.

The results are set forth in Table 3.

                  TABLE 3                                                         ______________________________________                                                  nmol/ml (Stimulation index)                                         Dose (μg/ml)                                                                           Ex.1       Ex.2       Ex.3                                        ______________________________________                                        0 (control) 20         20         20                                           0.01       43 (2.2)   85 (4.3)   50 (2.5)                                    0.1         59 (3.0)   61 (3.1)   60 (3.0)                                    1.0         77 (3.9)   66 (3.3)   66 (3.3)                                    10.0        57 (2.9)   79 (4.0)   88 (4.4)                                    ______________________________________                                    

The results obtained as nmol/ml and stimulation index with respect tothe control, show that the compounds of the invention stimulate thenitric oxide production at all the doses used. Particularly, thecompound of example 2 showed a stimulation peak already at 0.01 μg/ml(IS=4.3).

Effect on the leishmanicidal activity of murine peritoneal macrophage

The peritoneal cells collected as described above were seeded in 96-wellflat bottom plates (Nunc, Roskiled, DK) at a concentration of 10⁵ /100μl; and incubated per 24 hours at 37° C. At the end of the treatment,the cells not adherent to the well were separated and discharged,whereas the adherent cells were washed three time with culture mediumand incubated for 24 hours with the compounds in question, then thecells were infected through a 24 hours incubation with promastigotes ofLeishmania major (L. major, PVL49 strain supplied by Dr. Neal R. A.,London School of Hygiene and Tropical Medicine, London, UK). At the endof the infection treatment each well was added with 100 μl of a 0.01%solution od sodium dodecylsulphate in RPMI 1640, and the plates wereincubated for 30 minutes at 37° C. The Schneider's culture medium(Schneider Drosophila medium, Gibco Lab., Grand island, N.Y., USA) with30% of FCS was added. Each well was added with 1 μCi of ³ H-thymidine,and a 72 hours incubation was carried out at 37° C. The incorporation ofradioactivity by the living parasites inside the peritoneal cells, whichis correlated to the infection degree and consequently to theleishmanicidal activity of the cells, was measured by collecting thecells by means of a cell-harvester and measuring the radioactivity withliquid scintillation β-counter.

The results are set forth in Table 4.

                  TABLE 4                                                         ______________________________________                                                 cpm* (% reduciton of infection)**                                    Dose (μg/ml)                                                                          Es. 1       Es. 2     Es. 3                                        ______________________________________                                        (control)  18,273      18,273    18,273                                       0.1        5,096 (72)  17,568 (4)                                                                              18,826 (-3)                                  1.0        3,783 (79)  11,991 (34)                                                                             8,377 (54)                                   10.0       4,292 (77)  10,582 (42)                                                                             7,842 (57)                                   ______________________________________                                         *counts per minute                                                            ##STR3##                                                                 

The results obtained show that the compounds of the present inventionare able to reduce the infection degree. Particularly, the compound ofExample 1 showed to be the utmost effective, in fact it provided aninfection reduction of over 70% already with the minimum dose used inthe test.

Evaluation of the in vivo protection against septic shock

BALB/C mice (6 mice/group) weighting between 20 and 25 g, wereintraperitoneally inoculated with 50 mg/kg of LPS(lipopolysaccharide-Sigma). The groups, except for the control, wereinoculated with increasing doses of one of the compounds of theinvention dissolved in physiologic solution having a final volume of 0.5ml, at time 0 (i.e. together with LPS) or 30 minutes after the LPSinoculation. The mice were checked for 8 days to determined the survivalpercentage. Compound of Example 1 yields a survival percentage of about65% at doses of 6.2 ug/mouse and 0.62 ug/mouse, and its isomer B yieldsa survival percentage of about 80% at a dose of 6.25 ug/mouse and of 75%at a dose of 62.5 ug/mouse.

In view of what said, the compounds of the present invention are usefulin all the pathologic and non pathologic situations where it takes tostrenght or restore the immune response both in therapy and inprophylaxis. Therefore, as examples of use of the present compoundsthere may be cited bacteric infections, mainly in case of septic shock,viral infections (herpes), parasitosis, infections due to acquiredimmuno-efficiency syndrome, prophylaxis in youth and elder, heavilyburn, dialysis, tumours and transplantation. Moreover the compounds ofthe invention may be enployed as adjuvant in vaccines.

Also object of the present invention is the use of the new compounds asimmuno-stimulating agents, as well as to all the industrial aspectsconnected to said use, including the pharmaceutical compositionscontaining the compounds of the invention. For the intended therapeuticuses, the compounds of formula I may be administered suitably formulatedin pharmaceutical compositions according to what described, for example,in "Remington's Pharmaceutical Sciences Handbook", Mack Pub. Co., XVIIed., N.Y. USA. Obviously the posology will depend on several aspectssuch as the kind and severity of the case to be treated and theconditions of the patient (weight, age, sex, ecc.).

We claim:
 1. Retroinverted tetrapeptides of the general formula ##STR4##wherein R is a hydrogen atom or the side-chain of threonine; R₁ is theside-chain of arginine, leucine or glutamine; and R₂ is a hydrogen atomor a metabolically perishable acyl group; with the proviso that when R₁is the side-chain of arginine, R cannot be the side-chain of threonine;diastereoisomeric forms and pharmacologically acceptable salt, estersand amides thereof.
 2. Tetrapeptide according to claim 1 which isgGly-(R,S)mLys-Pro-Arg and the single diastereoisomeric forms. 3.Tetrapeptide according to claim 1 which is gThr-(R,S)mLys-Pro-Leu andthe single diastereoisomeric forms.
 4. Tetrapeptide according to claim 1which is gThr-(R,S)mLys-Pro-Gln and the single diastereoisomeric forms.5. A method for immunostimulation comprising administering to a subjectin need thereof an effective amount of a compound of formula (I)##STR5## wherein R is a hydrogen atom or the side-chain of threonine; R₁is the side-chain of arginine, leucine or glutamine; and R₂ is ahydrogen atom or a metabolically perishable acyl group; with the provisothat when R₁ is the side-chain of arginine, R cannot be the side-chainof threonine; diastereo-isomeric forms and pharmacologically acceptablesalts, esters and amides thereof.
 6. A pharmaceutical compositioncomprising an immuno-stimulating effective amount of at least onecompound of claim
 1. 7. The pharmaceutical composition of claim 6further comprising a pharmaceutically acceptable eccipient.